Anti-infectious carbohydrates

ABSTRACT

Neutral straight-chain or branched oligosaccharides for preventing the invasion and infection of mammal cells by pathogens and for fighting diseases caused by such pathogens are disclosed. Food, dietetic products and pharmaceutical agents containing oligosaccharides consist of a base unit of formula (I), and between 0 and 19 other units of formula (II), which are linked directly or indirectly to the base unit. Gal represents a galactose monosaccharide unit; Glc represents a glucose monosaccharide unit; HexNAc rpresents an N-acetylated galactosamine or glucosamine monosaccharide unit (GalNAc or GlcNAc); R independently represent a β 1-3 or β 1-6 glycosidic link to HexNAc monosaccharide of the next [Gal-HexNAc] unit (II), and on a terminal [Gal-HexNAc] unit, or R are not present; and a terminal [Gal-HexNAc] unit, wherein R represent a deoxyhexose radical, can have another deoxyhexose radical on the HexNAc monosaccharide unit.

DESCRIPTION

The invention relates to the use of neutral straight-chain or branchedoligosaccharides for preventing the invasion and infection of mammalcells by pathogens, and for combating diseases caused by such pathogens.The invention also relates to food, dietetic and pharmaceutical agentscontaining these oligosaccharides.

The adhesion of pathogenic organisms, as well as of cell-damagingsubstances to the surface of mammal cells is the first step and anessential prerequisite for an infection or impairment of the cell. Theinteraction between the pathogens and the cells comes about through aligand-receptor relationship. Glycostructures play an important role inthese relationships or interactions.

One possibility to influence such ligand-receptor relationships consistsin blocking and/or structurally changing the respective receptors on thecell surface or of the ligands.

In specific test systems, various carbohydrate mixtures have proven tobe very effective in reducing or completely preventing the adhesion, forexample of micro-organisms to the cell surface, cf. Kunz, C.; Rudloff,S. Acta Paediatr. 1993, 82, 903-912. Other substances such as the Lewisstructures as carbohydrate ligands of selectines (adhesion proteins onendothelia and lymphocytes) modulate the interaction between thelymphocytes and the endothelium, for example, in the context of rolling,homing and the invasion with inflammatory processes (Norman, K. E.;Anderson, G. P.; Kolb, H. C.; Ley, K.; Ernst, B. Blood 1998, 91,475-483). A further important physiological role in connection withbasic cellular functions, as well as with specific functions such ascell adhesion, migration, chemotaxis, proliferation, apoptose, neuritegrowth, are fulfilled by galactose-recognizing lectines, calledgalectines (Cooper D N & Barondes S H, Glycobiology, 1999 9 (10)979-984). It could be shown for the nematode C. elegans that itsgalectine LEC-1 can bind various galactose-containing oligosaccharidederivatives with different specificity (Arata Y. Hirabayashi J. Kasai K,JBC, 2001:276, 5, 3068-3077). In a model test with mice, the lethalityof an experimental listeriose could be reduced using galactose-specificlectines (Stoffel, B., Beuth J., Pulverer G., Zentralbl. Bakteriol.,1996, 284:439-442). The adherence of micro-organisms to host cells,however, may also be the trigger of signal cascades both in theexogenous pathogens and in the endogenous cells.

Another possibility consists in achieving an influence on molecularprocesses on the molecular biological level. This may lead to, forinstance in the case of mammal cells, defence mechanisms beingtriggered, or, in the case of pathogenic micro-organisms, the expressionof virulence mechanisms (e.g. cutting off virulence genes in bacteria byblocking central regulators) being reduced or prevented. In this way,the expression of certain surface structures of pathogenic listeriabacteria that are responsible for the invasion in host cells, may besuccessfully inhibited by certain carbohydrates such as cellobiose (ParkS F, Kroll R H, Mol Microbiol, 1993 8:653:661; WO-A 94/02586).

It is the object of the present invention to demonstrate a way how, withthe aid of oligosaccharides, the invasion and infection of mammal cellsby pathogens may be reduced or prevented, and how diseases caused bysuch pathogens may be effectively combated.

This object is fulfilled by the teaching of the claims.

According to the invention, specific oligosaccharides are used toachieve the object of the invention. These oligosaccharides are referredto below as inventive oligosaccharides.

According to the invention, single inventive oligosaccharides may beused alone, or several inventive oligosaccharides combined. Moreover, itis possible to use an inventive oligosaccharide or several inventiveoligosaccharides, or indeed a combination of a number of inventiveoligosaccharides together with other carbohydrates not figuring amongthe inventive oligosaccharides, in the form of a carbohydrate mixture.

The inventive oligosaccharides have a base unit of the following generalformula I:

wherein Gal represents a galactose monosaccharide unit, Glc represents aglucose monosaccharide unit, and HexNAc represents an N-acylatedgalactosamine monosaccharide unit or glucosamine monosaccharide unit(which may also be abbreviated as GalNAc or GlcNAc).

The residues R in general formula I do not have to be present, with thehydrogen atom not being considered as a residue within the framework ofthe present documents. Simply structured inventive oligosaccharides arethus those having the following general formula[GaL-HexNaC] - - - Gal-Glc.

Lacto-N-tetraose (Gal β1-3GlcNAcβ 1-3Galβ1-4Glc, LNT) which is preferredaccording to the invention, counts among these oligosaccharides.

In these latter oligosaccharides, the [Gal-HexNAc] unit represents aterminal unit. Same can be linked to the galactose monosaccharide unit(Gal) with one deoxyhexose (preferably an α1-2 glycosidic bond) or withtwo deoxyhexoses. The residue R or the residues R in the general formulaI may thus represent in this case such a deoxyhexose residue that ispreferably a rhamnose and fucose residue.

The lacto-N-tetraose that is preferred according to the invention,counts among these latter compounds, which lacto-N-tetraose isderivatized in an α1-2 glycosidic linkage at the terminal galactosemonosaccharide unit of the [Gal-HexNAc] unit by means of fucose, in thiscase lacto-N-fucopentaose I (LNFP I).

The residues R of the basic unit of the general formula I may alsorepresent linkage points to further [Gal-HexNAc] units of the followinggeneral formula II:

The residues R of these [Gal-HexNAc] units of the general formula IIthat are directly bound to the base unit of the general formula I (oneor two residues R may be present per unit) may in turn represent linkagepoints to further [Gal-HexNAc] units of the general formula II, whichfurther units are then indirectly bound to the base unit of the generalformula I. Also the further [Gal-HexNAc] units of the general formula IImay again feature such linkage points, and so on, the [Gal-HexNAc] unitsbeing bound through a β 1-3 or β 1-6 glycosidic linkage. Among thoseoligosaccharides count, for example, those of the following generalformulae, to name just a few:

The residues R in these formulae have the meanings indicated above.Thus, for example, all of the residues R may not be present.Furthermore, one or both of the residues R, or all of the residues R mayrepresent a linkage to the next [Gal-HexNAc] unit, which in turn eachmay have one residue R or two residues R, or else do not have any suchresidue R. All in all, up to 20 [Gal-HexNAc] units, and hence 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 of suchunits may be present in the inventive oligosaccharides, with the firstsuch unit belonging to the base unit of the general formula I, and theup to 19 further units of this type corresponding to the general formulaII. The inventive oligosaccharides thus may represent straight-chain orbranched molecules.

Assuming for explanation purposes that in the above formulae (III) to(VII), none of the residues R represents a linkage to the next[Gal-HexNAc] unit, then the compounds of formulae (III) and (IV) eachhave only one terminal [Gal-HexNAc] unit, i.e. that shown furthest tothe left, whereas the other units are in the middle position. Thecompounds of formulae (V), (VI) and (VII) each have two terminal unitsof this type, with compound (V) also having two, and compound (VII)having one unit of the general formula II located in the middle. Inthese terminal units, the residues R may also represent a deoxyhexoseresidue. Thus, one, two, three (if present) or all of the terminal[Gal-HexNAc] units of the inventive oligo-saccharides may have onedeoxyhexose residue of this type or two residues of this type.

In addition, one or more (if present) terminal [Gal-HexNAc] unit(s), inwhich the residue(s) R represent(s) a deoxyhexose residue, may have afurther deoxyhexose residue at the HexNAc monosaccharide unit.

The inventive oligosaccharides preferably include those oligosaccharideswhich meet one, two, three or even all four of the following criteria:

-   -   the deoxyhexose residue, if present, of the galactose        monosaccharide unit of the terminal [Gal-HexNAc] unit(s) is        linked to it in an α1-2 glycosidic bond,    -   the deoxyhexose residue, if present, of the galactose        monosaccharide unit of the terminal [Gal-HexNAc] unit(s) is a        rhamnose and fucose residue,    -   the further deoxyhexose residue, if present, of the HexNAc        monosaccharide unit of the terminal [Gal-HexNAc] unit(s) is        linked to it in an α1-3 or α1-4 glycosidic bond,    -   the further deoxyhexose residue, if present, of the HexNAc        monosaccharide unit of the terminal [Gal-HexNAc] unit(s) is a        rhamnose and fucose residue.

The inventive oligosaccharides are, incidentally, known compounds. Thus,the oligosaccharides LNT and LNFP I used according to the invention,count among the oligosaccharides that are present in human milk.

Surprisingly, it has been found that the inventive oligosaccharides atleast reduce or even prevent the invasion and infection of mammal cells,and may be used for combating diseases caused by such pathogens. Thesepathogens include invasive Gram-positive and Gram-negative pathogenicbacteria, e.g. intracellular bacteria, in particular listeria bacteriaand pathogenic viruses, e.g. rotaviruses.

It has been found, for example, that the inventive oligosaccharides mayprevent the invasion and infection of mammal cells by listeria bacteria,in particular Listeria monocytogenes. The results of the studiesconducted clearly show that neither the process of phagocytosis as such,nor the replication of the ingested listeria bacteria taken isinhibited. The inventive oligosaccharide LNFP I, among others, turnedout to have a particularly strong inhibitory effect.

The pentasaccharide LNFP I thus showed a strong, yet dose-dependantinhibitory action against listeria bacteria. Constitutional isomercompounds to LNFP I, to which, for example, the oligosaccharides LNFP IIand LNFP III and LNFP V belong, in contrast did not shown any suchaction. Incidentally, the oligosaccharides LNFP II and LNFP III are,like LNFP I, also present in human milk.

The inventive oligosaccharides may not only be used as freeoligosaccharides (i.e. having a reducing end) but may also be usedimmobilized on or adsorbed in a carrier. This carrier may be apeptide/protein (e.g. BSA), a lipid (glycolipid, ceramide), a polymer ora bio-polymer (e.g. carbohydrate dendrimer, polysaccharide,polyacrylamide) or any other aglykone.

The inventive oligosaccharides, be they free oligosaccharides oroligo-saccharides bound on a carrier, may be incorporated into variousfoods, dietetic and pharmaceutical compositions. All of thesecompositions may be present in liquid or solid form. The term food usedherein includes not only the foodstuff itself, but also nutritionalsupplements, drinks, as well as foods including infant and babyformulae. The term baby formulae or infant formulae comprises allartificially produced foods, but not human milk. “Artificial” here meansthose foods that are produced from raw materials of vegetable and animalorigin, but not of human origin. These foods may be administered to ahuman or an animal in any desired manner. This also includes theadministration as tube feeding into the stomach.

The inventive oligosaccharides may, for example, be added as admixturesor additives to the following products, although this enumeration is notconclusive: milk and milk products, infant and baby formulae, chocolatebars, yoghurt drinks, cheese, sausage and meat products, anabolic food,probe food and products for pregnant women.

The inventive oligosaccharides may also be administered in the form of apharmaceutical composition alone or together with one or severaladditional active agent(s). They may, for example, be formulated as atablet/sachet. For the formulation of such pharmaceuticals, usualadjuvants, carriers, auxiliary agents, diluents, moisturizing agents,thickening agents, flavoring agents, sweetening agents, etc. may beused.

The pharmaceutical compositions may be administered in any usual way toa patient (i.e. human or animal). However, for the sake of conveniencethese are compositions suited for the oral, lingual, nasal, intestinal,bronchial, vaginal, topical (skin and mucosa) and per os administration,and are formulated according to the kind of administration.

The food, dietetic and pharmaceutical compositions containing at leastone inventive oligosaccharide may be used among other things for theprevention and treatment of infections of the gastrointestinal tract,e.g. in case of listerioses, of the blood system, the respiratorypassages, the urogenital tract, as well as the nasopharynx, and forprotecting endothelia, epithelia and mucosa. They may thus also beapplied topically to the skin or may also be used on mucous membranes.These mucous membranes include the nasal, intestinal, bronchial andvaginal mucous membranes. The inventive oligosaccharides may forinstance be added to a mouth wash. All age groups, ranging from new bornbabies up to senior citizens, may be mentioned as/are potential targetgroups for the inventive oligosaccharides. Particular fields ofapplication are the protection and treatment of pregnant women, the sickas well as debilitated and elderly people, for whom the prevention e.g.of a listeriosis is of a particular importance.

The inventive oligosaccharides and, for example LNFP I are preparedchemically, enzymatically or using a combination of these twotechnologies according to suitable known methods. This takes place in asystematic manner from monosaccharide components or by modifyingsuitable oligosaccharide raw materials. In the enzymatic syntheses, bothtransferases (Leloir or non-Leloir) and hydrolases (reverse hydrolysisor transglycosilation) are used. The enzymes, may in this case be bothfree as well as bound (for instance, membrane reactor), or may becovalently bound to a carrier (e.g. beads, chromatography material orfiltration membranes). It is also possible to use procaryont oreucaryont cells for the synthesis, provided these cells have thesuitable enzymes. For further details for preparing the inventiveoligosaccharides, reference is made for example to Carbohydrates inChemistry and Biology (editors Ernst, Hart, Sinay, Wiley VCH-Weinheim2000, Vol. I-IV).

Exemplary dietetics and pharmaceuticals containing the inventiveoligosaccharides are given below.

Example 1

For preparing sachets, in each case 100 mg LNFP I are mixed in a drystate with 990 mg of maltodextrine, and then are packed in sachets.These sachets are administered three times per day during meals.

Example 2

A known medicinal food (i.e. Milupa® HN 25, balanced diet) in the formof a bead product containing 18.8 g of protein, 8.6 g of fat, 62.8 g ofcarbohydrates, 3.3 g of minerals and vitamins, is admixed in apreparation known per se with LNFP I in such an amount that 50 mg ofLNFP I are contained in 100 g of the finished bead product.

For the preparation of a liquid medicinal food, 100 ml of the knownmedicinal food Milupa HN 25 liquid (2.3 g of protein, 1.6 g of fat, 8.5g of carbohydrates, 37 g of minerals and vitamins) are admixed with 7 mgof LNFP I.

Example 3

A product for pregnant women

An effervescent tablet (final weight 4.15 g) (Neovin® from Milupa) isprepared in a manner known per se by admixing 200 to 500 mg of LNFP I.One tablet per day is dissolved in 150 ml water and swallowed.

Example 4

A product for the elderly and debilitated persons

A balanced pulverized medicinal food (Dilsana® from Milupa) containing22.5 g of protein, 7.7 g of fat, 60.8 g of carbohydrates, 5.4 g ofminerals and vitamins is prepared in a manner known per se byincorporating 100 mg to 1000 mg LNFP I per 100 g of powder. Up to 3×50 gper day of the food are dissolved in 150 ml water and administered.

Example 5

Tea

100 g of an instant tea powder prepared in a usual manner are mixed with2 g of LNFP I. 3.8 g of tea powder are dissolved in 100 ml of hot water,and administered three times per day.

Example 6

A protein-adapted infant milk formula (Aptamil® from Milupa) containing11.8 g of protein, 56.9 g of carbohydrates, 24.9 g of fat, 2.5 g ofminerals and vitamins and 45 mg of taurine are prepared in the usualmanner in the form of a bead product, which is mixed with 100 mg to 1000mg of LNFP I per 100 g of infant milk formula.

1. A composition of neutral straight-chain or branched oligosaccharidesof a base unit of the following general formula I:

and 0 to 19 further units directly or indirectly linked thereto andhaving the following general formula II:

wherein Gal represents a galactose monosaccharide unit, Glc represents aglucose monosaccharide unit, HexNAc represents an N-acetylatedgalactosamine monosaccharide or glucosamine monosaccharide unit (GalNAcor GlcNAc), the residues R represent, or are not present, as the casemay be, in each case independently of each other, a β1-3 or β1-6glycosidic bond to the HexNAc monosaccharide unit of the next[Gal-HexNAc] unit of the general formula II, and, at a terminal[Gal-HexNAc] unit, a deoxyhexose residue linked to the galactosemonosaccharide unit of said terminal [Gal-HexNAc] unit, and a terminal[Gal-HexNAc] unit, wherein the residue(s) R represent(s) a deoxyhexoseresidue, at which the HexNAc monosaccharide unit may have a furtherdeoxyhexose residue, for reducing and preventing the invasion andinfection of mammal cells by pathogens and for combating diseases causedby such pathogens.
 2. A composition according to claim 1, characterizedin that the oligosaccharides correspond to the following generalformula:

wherein the residues R have the meanings indicated above.
 3. Acomposition according to claim 1, characterized in that theoligosaccharides meet at least one of the following criteria: thedeoxyhexose residue, if present, of the galactose monosaccharide unit ofthe terminal [Gal-HexNAc] unit is linked to it in an α1-2 glycosidicbond, the deoxyhexose residue, if present, of the galactosemonosaccharide unit of the terminal [Gal-HexNAc] unit is a rhamnose orfucose residue, the further deoxyhexose residue, if present, of theHexNAc monosaccharide unit of the terminal [Gal-HexNAc] unit is linkedto it in an α1-3 or αl-4 glycosidic bond, the further deoxyhexoseresidue, if present, of the HexNAc monosaccharide unit of the terminal[Gal-HexNAc] unit is a rhamnose or fucose residue.
 4. A compositionaccording to claim 1, characterized in that (Gal β1-3GlcNAc β 1-3Galβ1-4Glc, LNT) is used as the oligosaccharide, lacto-N-tetraose.
 5. Acomposition according to claim 4, characterized in that thelacto-N-tetraose is derivatized in an α1-2 glycosidic bond at theterminal galactose monosaccharide unit by means of fucose, and islacto-N-fucopentaose I (LNFP I).
 6. A composition according to claim 1,characterized in that the oligosaccharides are immobilized on oradsorbed in an inert carrier.
 7. A composition according to claim 6,characterized in that the carrier is a peptide, a protein, a lipid, apolymer or a bio-polymer.
 8. A composition according to claim 1,characterized in that the oligosaccharides are incorporated in a liquidor solid food (with the exception of human milk), dietetic compositionsor pharmaceutical compositions for administration to a human or ananimal, or serve in the production of such an agent.
 9. A compositionaccording to claim 8, characterized in that the pharmaceuticalcompositions is suitable for oral, lingual, nasal, bronchial, vaginal,topical (skin and mucosa) or per os administration, or foradministration by means of a probe into the stomach of a human or ananimal.
 10. A composition according to claim 1, characterized in thatthe oligosaccharides are administered in an amount of at least 1 mg perkg of body weight and per day to a human or an animal, but not in theform of human milk.
 11. A composition according to claim 1,characterized in that the pathogens are invasive Gram-positive andGram-negative pathogenic bacteria, in particular listeria bacteria, andpathogenic viruses.
 12. A food, dietetic and pharmaceutical compositionscontaining at least one oligosaccharide described in claim 1, whereinthe pharmaceutical compositions in addition may contain a usualauxiliary agent or several usual auxiliary agents, including diluents,moisturizing agents, thickening agents, flavoring agents, sweeteningagents and carriers, as well as one further active agent or severalfurther active agents.
 13. A method of reducing or preventing theinvasion and infection of mammal cells by pathogens, and of combatingdiseases in humans and animals caused by such pathogens; characterizedin that at least one oligosaccharide, or a composition according toclaim 10 is administered, but not in the form of human milk, to a humanor an animal, in particular in an amount that at least 1 mg ofoligosaccharide per kg of body weight and per day is given to the humanor the animal.
 14. A method of reducing or preventing the invasion andinfection of mammal cells by pathogens, and of combating diseases inhumans and animals caused by such pathogens; characterized in that atleast one oligosaccharide, or a composition according to claim 11 isadministered, but not in the form of human milk, to a human or ananimal, in particular in an amount that at least 1 mg of oligosaccharideper kg of body weight and per day is given to the human or the animal